Cardiovascular disease is the leading cause of morbidity and mortality in the United States and in our veteran population. During the last funding cycle, we have identified several atrial-specifi ion channels including Cav1.3 (1D) L-type Ca2+ channel and small conductance Ca2+-activated K+ channels (SK or KCa2 channels) which play critical roles in the function of atrial myocytes as well as sinoatrial (SA) and atrioventricular (AV) nodes. Of clinical importance, we have demonstrated that SK channels are expressed and contribute significantly to the repolarization process in human atrial myocytes. For the current competing renewal application, we will focus our effort on the subcellular regulation of Ca2+ channels in ventricular myocytes and pacemaking cells. Embedded in our findings and the proposed project are relevant paradigm shifts that may be exploited in developing specific drugs for the treatment of cardiac arrhythmias. Specifically, we will test the central hypothesis that there is isoform-specific differential regulation of L-type Ca2+ current in ventricular myocytes and pacemaking cells by distinct isoforms of adenylyl cyclases (ACs). We will utilize new emerging techniques of live-cell imaging coupled with fluorescence resonance energy transfer (FRET)-based cAMP and protein kinase A (PKA) sensors to directly decipher the distinct subcellular localization and activities of different isoforms of ACs not only in ventricular myocytes but also in pacemaking cells. Indeed, we will take advantage of multidisciplinary techniques including in vivo and in vitro electrophysiologic recordings, live-cell imaging, and molecular modeling to determine the subcellular regulation of Ca2+ channels through distinct isoforms of ACs. Our proposed studies will expand our understanding of the specific subcellular localization and regulation of individual Ca2+ channels and how they might coordinate to mediate normal cardiac rhythm in vivo. Understanding the molecular and subcellular regulation of Ca2+ channels in the heart will set the stage for a new and more mechanistic approach for the treatment of cardiac arrhythmias and SA and AV node dysfunction, a common problem encountered in our veteran population.